The overall objective of this application is to refocus the first application's aims into one area, namely, the study of the role of the extracellular matrix synthesized by vascular endothelial cells (EC) in the inflammatory process. The proposed studies will follow-up on accomplishments realized since the first and then since the previous applications were submitted. Inflammation constitutes an important process of a variety of chronic diseases, including rheumatoid arthritis, skin diseases and lung fibrosis. Our studies show that basement membrane (BM) type IV collagen (Col IV) inhibits the activation of polymorphonuclear leukocytes (PMN's) by ligands such as N-f-Met-Leu-Phe, phorbol myristate acetate (PMA) or collagen type I. The inhibitory activity has been localized to the non-collagenous domain 1 (NC1) of Col IV and specifically to the NC1 domain of the alpha2(IV) chain. EHS tumor Col IV, which lacks the alpha3(IV) chain, does not inhibit PMN activation. Studies with synthetic peptides have shown that this property resides in a 19 amino acid sequence of the NC1 domain of the alpha3(IV) chain. We propose that during inflammation, EC and BM macromolecules play an active role in chemotaxis of PMN's, allowing their adhesion and diapedesis across the BM. In doing so Col IV displays a self-protective mechanism by inhibiting the production of superoxide (O2- ) and release of lysozomal enzymes, such as elastase and type IV collagenase. The proposed studies will aim at understanding (a) the structure-function relationships of peptides having the inhibitory property and (b) the mechanism(s) regulating the interaction of PMN with the NC1 domain of the alpha2(IV) chain. The specific aims of the proposal are to: (1) compare normal intact Col IV and its 7S and NC1 domains with the EHS Col IV and its 7S and NC1 domains in their ability to influence PMN functions such as O2- generation, PMN adhesion and chemotaxis. Comparable studies will be carried out using recombinant NC1 alpha1-alpha5(IV) chains; (2) study the chemotactic responsiveness of PMNs to N-f-Met-Leu-Phe and to leukotriene B4 (T4) in the presence of cAMP elevating normal Col IV, of the NC1 peptide alpha3(IV) 185-203 and the same peptide with specific amino acid substitutions. Determine the effect of inhibitors of adenylate cyclase and/or protein Kinase-A on cAMP levels in the presence of type IV collagen; (3) study the inhibitory activity of peptides arising from similar regions of NC1 domains of the alpha1, alpha2, alpha3, alpha4 and alpha5IV) chains; (4) examine the molecular mechanisms involved in the interaction between the NC1 domain of Col IV and PMN's. It is anticipated that the proposed studies would provide a better understanding of the role of the subendothelial BM in the inflammatory process.